This invention relates to methodology and an improved diluent system for use in the differentiation of leukocytes.
The use of instrumentation to count red blood cells and white blood cells has been available for close to 30 years. The earliest instruments and the most modern require a lysing agent which hemolyzes the red blood cells so that the white blood cells can be counted. A variety of lysing agents have been used which differ in their rate of hemolysis and their effect on the white blood cells.
A. Hatch and T. Balazs in the American Journal of Clinical Pathology, Vol. 36, No. 3, pp. 220-223, 1961, describe the use of acetyltrimethyl ammonium bromide, saponin and Triton.RTM. to lyse red blood cells and produce white blood cell (WBC) distribution. It is important that the authors noted that different sized leucocytes can be observed. They were able only to get a "rough estimate of the different types of white cells present". Their comments are of interest:
"Distribution of cell sizes were easily obtained but, inasmuch as there was a large amount of overlapping in size and type of cell they indicated only a rough estimate of the different types of white cells present. A differential stromatolysing agent would be required for a true differential picture. Akeroyd and associates report that the initial decrease in white cell counts using Triton.RTM. is owing to lysis of the lymphocytes. If this is true, it should be possible to perform differential blood counts on the electronic counter."
The authors clearly saw the need for adjustment of the lysing agent to obtain several populations of white cells.
Several years later in 1974, Hughes-Jones et al described in the Journal of Clinical Pathology, Vol. 27, pp. 623-625, preparation of an analysis system that distinguished neutrophiles and lymphocytes in blood. They used saponin to lyse the red cells and to separate the WBC but pointed out the difficult of adjusting the concentration of lysing agent.
P. A. Wycherley and M. J. O'Shea in the Journal of Clinical Pathology, Vol. 31, pp. 271-274, 1978 reported a further development of the method by linking a Coulter Channelyzer.RTM. to a Coulter Model S.RTM. cell counter. They were able to standardize the lysing solution and the method of analysis. The extension of the earlier studies to include a third population of cells was reported by Ledis et al in U.S. Pat. No. 4,485,175 where they used a mixture of alkyl ammonium chloride surfactants and added them slowly, and at a dilute concentration, to lyse the red cells and produce WBC separation. Procaine is added to stabilize the white cells. The authors point out that the quaternary ammonium compounds vary in composition and purity and, therefore, that it is necessary to adjust the concentration of lysing agent to accommodate this problem.
Matsuda and Shinkai in U.S. Pat. No. 4,656,139 reported a similar system using quaternary ammonium lysing agents with citric acid and a diluent containing a boric acid buffer. The citric acid shrinks the red cell ghosts so that they do not interfere with the WBC count.
In U.S. Pat. No. 4,745,071 a single quaternary ammonium surfactant is used instead of a mixture. The use of dodecyltrimethyl ammonium chloride is claimed to give the gentle red cell lysis required, and provides a uniquely selective effect on the leucocytes. This surfactant was previously used in admixture with other quaternary ammonium surfactants.
The only method with a considerably different approach is that described by Brown and Kirchanski in U.S. Pat. No. 4,637,986. According to this method, an organic buffer and leukoprotective agents at a pH of 8.5 are employed to produce a differential white cell count.
The above investigations show only slight differences in approach. All of them indicate the need for a surfactant, usually a quaternary ammonium salt, and the careful adjustment of the concentration. This careful adjustment was evident to the early investigators, and this is necessary because variation in strength and purity of the agents affects the lysing action of the surfactant.
Needless to say, the search for a red blood cell lysing procedure for separating and counting white blood cells which removes the necessity of careful adjustments continues.
It is an object of the invention to provide an improved method for differentiating leukocytes (white blood cells) in whole blood by the lysing of red blood cells therein which method does not require careful adjustments.
It is also an object of the invention to provide a more reliable method for the lysis of red blood cells for differentiation of white blood cells.
Yet another object of the invention is to provide a novel method of stabilizing white blood cells during the lysis of blood cells.
A further object of the invention is to provide a novel diluent for use in diluting whole blood samples to be mixed with lysing agents for the volumetric differentiation of white blood cells.
Another object of the invention is to provide novel reagents which make it possible to use a single diluent and lysing agent in most of the differential instruments available (e.g., Coulter.RTM. S Series, Sequoia-Turner.RTM. 2000, 1600, 1500 and Baker.RTM. 900).